Rejuvenating effects of young extracellular vesicles in aged rats and in cellular models of human senescence

Rejuvenation of an old organism was achieved in heterochronic parabiosis experiments, implicating different soluble factors in this effect. Extracellular vesicles (EVs) are the secretory effectors of many cells, including cardiosphere-derived cells (CDCs) with demonstrated anti-senescent effect. 1. To determine the role of EVs (versus other blood fractions) on the rejuvenating effect of the young blood. 2. To evaluate the anti-aging properties of therapeutically administered EVs secreted by young-CDCs in an old organism. Neonatal blood fractioned in 4 components (whole blood, serum, EV-depleted serum and purified EVs) was used to treat old human cardiac stromal cells (CSPCs). CDCs were generated from neonatal rat hearts and the secreted CDC-EVs were purified. CDC-EVs were then tested in naturally-aged rats, using monthly injections over 4-months period. For validation in human samples, pediatric CDC-EVs were tested in aged human CSPCs and progeric fibroblasts. While the purified EVs reproduced the rejuvenating effects of the whole blood, CSPCs treated with EV-depleted serum exhibited the highest degree of senescence. Treatment with young CDC-EVs induce structural and functional improvements in the heart, lungs, skeletal muscle, and kidneys of old rats, while favorably modulating glucose metabolism and anti-senescence pathways. Lifespan was prolonged. EVs secreted by young CDCs exert broad-ranging anti-aging effects in aged rodents and in cellular models of human senescence. Our work not only identifies CDC-EVs as possible therapeutic candidates for a wide range of age-related pathologies, but also raises the question of whether EVs function as endogenous modulators of senescence.

www.nature.com/scientificreports/ Here, we address the following questions: Do CDC-EVs exert anti-aging properties in vivo? Are favorable changes durable, and do CDC-EVs affect longevity? Finally, do EVs (versus other blood fractions) mediate antisenescent effects of young blood observed in heterochronic parabiosis 12 ?

Methods
For detailed methods, please see the supplementary file.
In vivo experiments. Twenty-two-month-old Fisher 344 rats (male and female) were obtained from the National Institute of Aging. All methods were carried out in accordance with relevant guidelines, approved by the  23, revised 1996), and are reported in accordance with ARRIVE guidelines. Old Fisher 344 rats were selected for this study since they mimic different human aging-related conditions such as cardiac fibrosis and diastolic dysfunction 10,13 . After initial evaluation with echocardiography and exercise testing, a total of 27 animals were divided randomly into two groups 1 : 13 rats treated with extracellular vesicles secreted by neonatal cardiosphere-derived cells (CDC-EVs) 2

Results
CDC-EVs were isolated from neonatal rat CDCs as described in Methods and Fig. 1A. CDC-EVs resuspended in PBS, or PBS alone, were injected percutaneously under ultrasound guidance into the LV lumen of 22-monthold Fisher 344 rats ("old rats") on a monthly basis, to achieve systemic arterial delivery. Animals were followed for 16 weeks and survivors were euthanized for further studies.
CDC-EV effects in aged hearts: from tissue rejuvenation to functional improvement. After 4 monthly systemic injections, the hearts of CDC-EV-treated rats, but not control rats, showed signs of rejuvenation: telomere length (~two-fold increase, p < 0.001; Fig. 1B,C), phosphorylated histone H2A (γH2AX; 32% decrease, p < 0.001; Fig. 1D,E, Suppl Figure 2A), and interleukin 6 (IL-6; 21% decrease, p = 0.03; Fig. 1F), a component of the senescence-associated secretory phenotype 14 , were all favorably modulated. Telomere length was Figure 1. Signs of heart rejuvenation in old rats treated with extracellular vesicles secreted by neonatal cardiosphere-derived cells (CDC-EVs). (A) A schematic of the main study protocol. Heart explant-derived cells from thirty F344 rat pups were pooled together to generate CDCs. CDC-EVs were purified from serum-free medium conditioned for 15 days by CDCs and resuspended in phosphate-buffered saline (PBS). 22-month-old F344 rats after an initial evaluation were randomized to receive percutaneous intra-left ventricular injection of CDC-EVs resuspended in PBS or PBS alone (300 μL). A higher "loading" dose of CDC-EVs was followed by lower consecutive monthly doses. (B) Telomere length in heart cells. Representative detail of confocal maximum projection images of telomere Q-FISH (TEL-Cy3) and alpha-sarcomeric actinin (α-SA) immunofluorescence in old animals treated with PBS (n = 6) and CDC-EVs (n = 5). www.nature.com/scientificreports/ 2 times longer in CDC-EV treated versus control rats, as in our previous study using CDCs 10 . Ingenuity pathway analysis of the transcriptomes from CDC-EV and control rat hearts (Fig. 1G) identified changes in 3 major upstream regulators ( Fig. 1H and Suppl Figure 2B): NANOG, a pluripotency marker and cell reprogramming enhancer (z = 2; p = 0.001) 15,16 ; immune B-cell receptor complex (BCR, z = 2; p < 0.001); and extracellular signalregulated protein kinases 1 and 2 (ERK1/2, z = 2.6; p < 0.001). In the serum of CDC-EV treated rats, we observed an increase in alkaline phosphatase (34 IU/L, p = 0.01) and preservation of globulin levels compared with controls (Fig. 1J). These may be associated with an increase in circulating stem/progenitor cells 17 and preservation of immunity 18 in CDC-EV-treated rats, respectively, but we have not tested these conjectures.
Both serial echocardiography and endpoint invasive hemodynamics revealed reduced LV stiffness (i.e., improvement of diastolic function) in rats treated with CDC-EVs. Echocardiography showed progressive changes of E′ and E/E′ ratio, with improvements in the CDC-EV group but deterioration in controls (E'-wave 11.2 ± 1.4 mm/s increase; p = 0.02 in CDC-EV group and 10.9 ± 12.8 mm/s decrease; p = 0.06 in PBS group at study end-point; Fig. 3A, B; Suppl. Figure 3). Elastance catheter analysis showed decreases in the slopes of the end-diastolic pressure-volume relationship in CDC-EV rats, but not controls (EDPVR; 50% decrease, p = 0.02;

Metabolic effects of CDC-EVs in old rats.
Rats injected with CDC-EVs lost weight over the 16 weeks of follow up (Suppl. Figure 4A), in association with decreased visceral abdominal fat on necropsy (Suppl. Figure 4B). Blood glucose levels tended to fall in CDC-EV rats, while the opposite tendency was seen in controls (Suppl. Figure 4C). Analysis of several proteins implicated in insulin sensitivity and oxidative metabolism in biceps femoris lysates revealed up-regulation in CDC-EV-rats versus controls (1.7-fold up-regulation of citrate synthase activity, p < 0.001; Suppl. Figure 4D, E, Suppl. Figure 8), consistent with higher mitochondrial biogenesis 20 . To assay the effects of CDC-EVs on glucose metabolism, we performed glucose tolerance tests at baseline and 4 weeks later (end-point) in a separate group of 22-month-old rats. The end-point test was done 48 h after CDC-EV or PBS infusion (Suppl. Figure 4F). In PBS-infused controls, the basal and follow-up responses of blood glucose were comparable, but glucose levels were significantly lower just 2 days after CDC-EV infusion (Suppl. Figure 4G). The reductions in visceral fat and the improved glucose tolerance support the idea that CDC-EVs may decrease age-related insulin resistance. In support of this hypothesis, CDC-EV rats displayed lower levels of fasting serum insulin and greater hyperglycemia-induced insulin secretion than controls (Suppl. Figure 4H).
Other systemic effects of CDC-EVs in aged rats. Exercise capacity is increased in aged rats after infusion of neonatal CDCs 10 . Likewise, CDC-EVs increased treadmill exercise capacity by ~16% in the month after the first dose (133 ± 51 m to 154 ± 61 m; p < 0.05) and remained higher than in the PBS group (which deteriorated progressively) throughout follow-up (Fig. 4A). Moreover, survival was improved in CDC-EV-treated rats versus PBS controls, with the latency to death increased by 54 days (Fig. 4B), which extrapolates to ~3.2 human years 21 . Spontaneous leukemia is a frequent cause of death in aged F344 rats 22 . Indeed, leukemia-related death occurred in 50% of PBS-injected rats versus 28% in the CDC-EV-injected animals, consistent with previous findings 11 .
Aging causes progressive replacement of healthy parenchyma by fibrotic tissue in a variety of organs, contributing to loss of function 23 . The heart, skeletal muscle, and lungs exhibited extensive fibrosis in the control rats (~8%) but decreased strikingly (to ~30% of control levels) in CDC-EV animals ( Fig. 4C-G). Similarly, the kidneys of aged control rats had appreciable glomerulosclerosis, which is a common cause of renal dysfunction in old F344 rats 24 . The number of sclerotic glomeruli was lower in CDC-EV vs PBS-injected rats (Fig. 4G,H), rationalizing the improvements in circulating biomarkers of renal function in the CDC-EV group (Suppl. Figure 5).

Anti-senescent effects of CDC-EVs are reproduced in aged human heart cells and progeric fibroblasts.
To exclude rodent-specific effects, we tested rejuvenating effects of CDC-EVs from young human (donors < 2 years of age) on aged human cells. In vitro assays used cardiac stromal/progenitor cells (CSPC) obtained from > 55-year-old human donors and dermal fibroblasts from progeric patients. Aged human cells were treated with CDC-EVs resuspended in serum-free conditioned medium or serum-free medium alone (Methods).
Exposure of aged CSPC with young CDC-EVs triggered favorable modulation of DNA repair genes (Suppl. Figure 6A), down-regulation of protein effectors of the chronic senescence pathway 4,25 and an increase in antioxidative proteins 26,27 (Suppl. Figure 6B). Transcriptional and proteomic changes observed in CDC-EV primed CSPC versus control cells were associated with increased proliferation, decreased apoptosis (Suppl. Figure 6C, D, respectively), and increased self-assembly potential (Suppl. Figure 6E). The benefits were not limited to cells of cardiovascular origin: CDC-EV-primed progeric fibroblasts manifested rejuvenation in terms of increased proliferation, decreased number of senescent cells (Suppl. Figure 6F, G, respectively), and favorable changes in the transcriptome (Suppl. Figure 7).    www.nature.com/scientificreports/ GDF11 as an anti-senescent mediator 28,29 (conclusions which were later disputed 30,31 ;). Since EVs are the soluble effectors of the CDCs 32 , we decided to compare their anti-senescent properties relative to those of other blood fractions in vitro, simulating heterochronic parabiosis model (Methods, Fig. 5A-C). Here, old human donor CSPCs were treated with different fractions of neonatal rat blood. After 48 h' incubation, CSPCs displayed two indicators of cellular aging (senescence-associated β galactosidase (SA-β-GAL), γH2AX, a DNA damage response marker), and diminished proliferative activity (as reported by Ki-67). Exposure to whole blood or serum decreased SA-β-GAL and γH2AX, while increasing Ki67 positivity. While the EV fraction reproduced these effects, cells treated with EV-depleted serum exhibited the highest degree of senescence, and the least cellular proliferation (Fig. 5D,E). The differences appear to be independent of circulating proteins, insofar as the free protein concentration in the EV-depleted-serum fraction was unchanged relative to total serum, but the concentration of EVs was ~10-times lower. These results implicate EVs as circulating bloodborne endogenous modulators of senescence.

Discussion
Repeated systemic administration of young CDC-EVs in aged rodents triggered broad-ranging functional improvements, with concordant structural changes in different organs and associated evidence of tissue rejuvenation (Fig. 6). The beneficial effects of CDC-EVs were maintained over mid-term follow-up, with prolongation of survival of treated animals. But, beyond longevity, the changes we observed in heart and kidney function, glucose metabolism, and exercise tolerance have the potential to improve quality of life, which is an important goal of anti-aging therapies 33 . Cardiovascular diseases, diabetes, and cancer are aging-related conditions which underlie much morbidity and mortality in the elderly population 1,2,4 . Using a single cell-free therapeutic agent, young CDC-EVs, we demonstrated that all three pathologies can be favorably modulated. Moreover, tissue fibrosis contributing to organ dysfunction 23,34 was broadly ameliorated (heart, lungs, skeletal muscle and kidneys exhibited less interstitial fibrosis) in CDC-EV treated rats. Based on these findings, CDC-EVs emerge as a strategy capable of targeting pathophysiologic mechanism(s) underlying many age-related chronic conditions. Both, MiR-146 and miR-92a highly enriched in CDC-EVs 11,32 known to be implicated in aging-related pathways 35,36 may have played a role in rejuvenating effects observed in our study.
Cellular senescence is thought to contribute to progressive age-related organ dysfunction 4,25 . Senescent cells are characterized by extensive transcriptional changes, telomere attrition, chronically stimulated DNA damage response, mitochondrial dysfunction, the senescence-associated secretory phenotype (SASP), and cell-cycle arrest [37][38][39] . Previously, we described an anti-senescent effect of young CDC-EVs in vitro 10 . Here, we confirm that cellular rejuvenation, conceived as partial or total reversal of senescence, can be also achieved in vivo in old animals injected with young CDC-EVs. Benefits include telomere elongation in heart cells, less-active DNA damage response (represented by phosphorylated γH2AX, 40 ), lower IL-6 levels, and changes in protein levels suggestive of enhanced mitochondrial biogenesis in skeletal muscle. Extensive transcriptomic differences in treated versus control groups were consistent with the observed upregulation of the transcription factor NANOG and extracellular signal-regulated kinase ERK 1/2. Both are recognized regulators and stabilizers of the pluripotency gene regulatory network 15 . Accordingly, we speculate that the mechanism of action of young CDC-EVs is related in part to the control of the dynamic state of pluripotency and reprogramming 41 , a strategy that has been touted in pursuit of rejuvenation 42 . This idea is also supported by the increased self-assembly potential of young CDC-EV-primed senescent human heart cells in vitro.
Among the various approaches being tested to promote rejuvenation [42][43][44][45] , the use of young blood or its components has gained particular traction, including trials in humans ( 28,46,47 , ClinicalTrials.gov Identifier: NCT02803554). Much effort has been put into identifying the blood component(s) responsible for the rejuvenating effects 47,48 , without conclusive results to date. While acknowledging the limitations of our in vitro "parabiosis" experiment, we observed that EVs are required for the rejuvenating effects of neonatal serum. In fact, treatment with neonatal CDC-EVs in old animals, and in vitro on senescent human cells, induced multiple broad anti-aging effects.
Several limitations of our study need to be considered when interpreting the results. Although we selected a naturally-aged rat model, rodents may do not fully recapitulate the aging process in larger mammals. We did, however, confirm the rejuvenating effects of CDC-EVs in models of human cellular senescence (CSPCs from aged donors, and dermal fibroblasts from progeric patients) in vitro. Additionally, the functional studies were performed sequentially, allowing us to evaluate CDC-EV-induced changes in the same animal, but histopathological evaluation could only be performed at study end-point, so only the differences between groups at that specific time point were assessed. In the in vitro experiment simulating parabiosis, although the EV fraction was purified and separated from free proteins by the density gradient method, we cannot rule out that some individual proteins may be different quantitatively in the EV-depleted serum fraction. Another limitation of the simulated in vitro parabiosis was the lack of true cross-circulation; however, it would have been impossible experimentally, using currently available methods, to deplete EVs selectively in vivo in one paired animal and not the other.
Translational relevance. Aging-related diastolic dysfunction and the associated heart failure represent unmet clinical needs. Since classical therapeutic targets pursued in systolic heart failure have failed, novel biological interventions are being explored. Here, we find that EVs secreted by young donors' cells can reverse various aging-related pathologies in senescent rats. Infusion of young CDC-EVs trigger systemic rejuvenation as manifested by improved glucose metabolism, exercise capacity, and survival. In the heart, signs of cellular rejuvenation were associated with structural (less fibrosis and hypertrophy) and functional (decreased stiffness) improvements. Rejuvenating effects of young CDC-EVs were validated in human models of cellular senescence. www.nature.com/scientificreports/ Given that allogeneic CDCs are already in advanced testing and have proven safe to date, such cells can be used as manufacturing platforms for EVs, enabling rapid progress to clinical testing in a variety of aging-related disorders.
In conclusion, EVs are nanoparticles containing thousands of known and potential signaling molecules (ribonucleic acids, proteins, lipids) 32,49 . EVs can influence multiple pathways synergistically; indeed, our findings indicate or hint that CDC-EVs modulate not only the telomere-telomerase axis 10 and the p53-p38-MAPK pathway, but also the DNA damage response, oxidative stress, mitochondrial function, and pluripotency dynamics. Even more processes, yet to be explored, may underlie the observed benefits on lifespan, metabolic health, tissue composition and cardiac function.

Data availability
All data generated or analysed during this study are included in this published article and its supplementary information files.
Received: 11 December 2022; Accepted: 25 July 2023 Figure 6. Extracellular vesicles secreted by young cardiac progenitor cells exert broad-ranging anti-aging effects in naturally-aged animals. Given the heterogeneous bioactive content of the vesicles modulation of multiple but synchronized pathways, related with the antisenescence mechanism of action, underlain the observed structural and functional changes.